Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

University of Minnesota

To document an improvement in the quality of life in a group of patients with refractory angina and videothoracoscopic sympathectomy (VTSY) during the early postoperative period and a six-month follow-up.

Endoscopic thoracic sympathectomy--its effect in the treatment of refractory angina pectoris.

We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. A shotgun genomic library was screened with genomic DNA to isolate and characterize the most abundant elements. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. We also amplified transposable elements with degenerate primers and used them to screen the library. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Other types of elements were ubiquitous on all chromosomes. Moreover, we isolated and characterized two new tandem repeats. One of them, STAR-C, was localized at the centromeres of all chromosomes except the Y chromosome, where it was present on the p-arm. Its variant, STAR-Y, carrying a small deletion, was specifically localized on the q-arm of the Y chromosome. The second tandem repeat, TR1, co-localized with the 45S rDNA cluster in the subtelomeres of five pairs of autosomes. FISH analysis of other Silene species revealed that some elements (e.g., Ogre-like elements) are confined to the section Elisanthe while others (e.g. Copia or Athila-like elements) are present also in more distant species. Similarly, the centromeric satellite STAR-C was conserved in the genus Silene whereas the subtelomeric satellite X-43.1 was specific for Elisanthe section. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements. The unique pattern of repeat distribution is found on the Y chromosome, where some elements are accumulated while other elements are conspicuously absent, which probably reflects different forces shaping the Y chromosome.

Survey of repetitive sequences in Silene latifolia with respect to their distribution on sex chromosomes.

Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

Targeting DNA double-strand breaks with TAL effector nucleases.

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.

DNA replicons for plant genome engineering.

Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed.

Tomas Cermak

Department of Genetics,
Cell Biology & Development and Center for Genome Engineering,
Department of Genetics, Cell Biology & Development and Center for Genome Engineering

University of Minnesota

Mouse Genome Engineering Using Designer Nucleases

Tomas Cermak

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics,Cell Biology & Development and Center for Genome Engineering,Department of Genetics, Cell Biology & Development and Center for Genome Engineering

University of Minnesota

Mouse Genome Engineering Using Designer Nucleases

Department of Genetics,
Cell Biology & Development and Center for Genome Engineering,
Department of Genetics, Cell Biology & Development and Center for Genome Engineering